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Ultrastructural Analysis of Vesicular Transport in Electrotransfection

Published online by Cambridge University Press:  18 October 2018

Liangli Wang ,
Sara E. Miller  and
Fan Yuan
Liangli Wang
Affiliation:
Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA
Sara E. Miller*
Affiliation:
Department of Pathology, Duke University Medical School, Durham, NC 27710, USA
Fan Yuan*
Affiliation:
Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA
*
*Authors for correspondence: Sara E. Miller, E-mail: mille012@duke.edu; Fan Yuan, E-mail: fyuan@duke.edu
*Authors for correspondence: Sara E. Miller, E-mail: mille012@duke.edu; Fan Yuan, E-mail: fyuan@duke.edu
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Abstract

Emerging evidence from various studies indicates that plasmid DNA (pDNA) is internalized by cells through an endocytosis-like process when it is used for electrotransfection. To provide morphological evidence of the process, we investigated ultrastructures in cells that were associated with the electrotransfected pDNA, using immunoelectron microscopy. The results demonstrate that four endocytic pathways are involved in the uptake of the pDNA, including caveolae- and clathrin-mediated endocytosis, macropinocytosis, and the clathrin-independent carrier/glycosylphosphatidylinositol-anchored protein-enriched early endosomal compartment (CLIC/GEEC) pathway. Among them, macropinocytosis is the most common pathway utilized by cells having various pDNA uptake capacities, and the CLIC/GEEC pathway is observed primarily in human umbilical vein endothelial cells. Quantitatively, the endocytic pathways are more active in easy-to-transfect cells than in hard-to-transfect ones. Taken together, our data provide ultrastructural evidence showing that endocytosis plays an important role in cellular uptake and intracellular transport of electrotransfected pDNA.

Keywords

electrotransfectionelectroporationintracellular traffickingclathrin-mediated endocytosismacropinocytosis
Type
Biological Science Applications
Information
Microscopy and Microanalysis , Volume 24 , Issue 5 , October 2018 , pp. 553 - 563
Copyright
© Microscopy Society of America 2018 

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References

Agarwal, A, Zudans, I, Weber, EA, Olofsson, J, Orwar, O Weber, SG (2007) Effect of cell size and shape on single-cell electroporation. Anal Chem 79(10), 3589–3596.Google Scholar
Boulant, S, Kural, C, Zeeh, JC, Ubelmann, F Kirchhausen, T (2011) Actin dynamics counteract membrane tension during clathrin-mediated endocytosis. Nat Cell Biol 13(9), 1124–1131.Google Scholar
Bretscher, MS, Thomson, JN Pearse, BM (1980) Coated pits act as molecular filters. Proc Natl Acad Sci USA 77(7), 4156–4159.Google Scholar
Bruns, RR Palade, GE (1968a) Studies on blood capillaries. I. General organization of blood capillaries in muscle. J Cell Biol 37(2), 244–276.Google Scholar
Bruns, RR Palade, GE (1968b) Studies on blood capillaries. II. Transport of ferritin molecules across the wall of muscle capillaries. . J Cell Biol 37(2), 277–299.Google Scholar
Cervia, LD, Chang, CC, Wang, L Yuan, F (2017) Distinct effects of endosomal escape and inhibition of endosomal trafficking on gene delivery via electrotransfection. PLoS One 12(2), e0171699.Google Scholar
Cervia, LD Yuan, F (2018) Current progress in electrotransfection as a nonviral method for gene delivery. Mol Pharm 15(9), 3617–3624.Google Scholar
Chang, CC, Wu, M Yuan, F (2014) Role of specific endocytic pathways in electrotransfection of cells. Mol Ther Methods Clin Dev 1, 14058.Google Scholar
Chernomordik, LV, Sokolov, AV Budker, VG (1990) Electrostimulated uptake of DNA by liposomes. Biochim Biophys Acta 1024(1), 179–183.Google Scholar
de Gennes, PG (1999) Passive entry of a DNA molecule into a small pore. Proc Natl Acad Sci USA 96(13), 7262–7264.Google Scholar
Doherty, GJ McMahon, HT (2009) Mechanisms of endocytosis. Annu Rev Biochem 78, 857–902.Google Scholar
El-Sayed, A Harashima, H (2013) Endocytosis of gene delivery vectors: from clathrin-dependent to lipid raft-mediated endocytosis. Mol Ther 21(6), 1118–1130.Google Scholar
Escoffre, JM, Portet, T, Wasungu, L, Teissie, J, Dean, D Rols, MP (2009) What is (still not) known of the mechanism by which electroporation mediates gene transfer and expression in cells and tissues. Mol Biotechnol 41(3), 286–295.Google Scholar
Eynard, N, Rols, MP, Ganeva, V, Galutzov, B, Sabri, N Teissie, J (1997) Electrotransformation pathways of procaryotic and eucaryotic cells: recent developments. Bioelectrochem Bioenerg 44(1), 103–110.Google Scholar
Golzio, M, Teissie, J Rols, MP (2002) Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci USA 99(3), 1292–1297.Google Scholar
Hansen, CG Nichols, BJ (2009) Molecular mechanisms of clathrin-independent endocytosis. J Cell Sci 122(Pt 11), 1713–1721.Google Scholar
Heller, LC Heller, R (2006) In vivo electroporation for gene therapy. Hum Gene Ther 17(9), 890–897.Google Scholar
Heller, R Heller, LC (2015) Gene electrotransfer clinical trials. Adv Genet 89, 235–262.Google Scholar
Henshaw, J, Mossop, B Yuan, F (2008) Relaxin treatment of solid tumors: effects on electric field-mediated gene delivery. Mol Cancer Ther 7(8), 2566–2573.Google Scholar
Henshaw, J, Mossop, B Yuan, F (2011) Enhancement of electric field-mediated gene delivery through pretreatment of tumors with a hyperosmotic mannitol solution. Cancer Gene Ther 18(1), 26–33.Google Scholar
Henshaw, JW Yuan, F (2008) Field distribution and DNA transport in solid tumors during electric field-mediated gene delivery. J Pharm Sci 97(2), 691–711.Google Scholar
Howes, MT, Kirkham, M, Riches, J, Cortese, K, Walser, PJ, Simpson, F, Hill, MM, Jones, A, Lundmark, R, Lindsay, MR, Hernandez-Deviez, DJ, Hadzic, G, McCluskey, A, Bashir, R, Liu, L, Pilch, P, McMahon, H, Robinson, PJ, Hancock, JF, Mayor, S Parton, RG (2010) Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells. J Cell Biol 190(4), 675–691.Google Scholar
Hristova, NI, Tsoneva, I Neumann, E (1997) Sphingosine-mediated electroporative DNA transfer through lipid bilayers. FEBS Lett 415(1), 81–86.Google Scholar
Hunt, MA, Currie, MJ, Robinson, BA Dachs, GU (2010) Optimizing transfection of primary human umbilical vein endothelial cells using commercially available chemical transfection reagents. J Biomol Tech 21(2), 66–72.Google Scholar
Huotari, J Helenius, A (2011) Endosome maturation. EMBO J 30(17), 3481–3500.Google Scholar
Jiang, R, Gao, B, Prasad, K, Greene, LE Eisenberg, E (2000) Hsc70 chaperones clathrin and primes it to interact with vesicle membranes. J Biol Chem 275(12), 8439–8447.Google Scholar
Jordan, ET, Collins, M, Terefe, J, Ugozzoli, L Rubio, T (2008) Optimizing electroporation conditions in primary and other difficult-to-transfect cells. J Biomol Tech 19(5), 328–334.Google Scholar
Klenchin, VA, Sukharev, SI, Serov, SM, Chernomordik, LV Chizmadzhev Yu, A (1991) Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis. Biophys J 60(4), 804–811.Google Scholar
Kotnik, T, Pucihar, G, Rebersek, M, Miklavcic, D Mir, LM (2003) Role of pulse shape in cell membrane electropermeabilization. Biochim Biophys Acta 1614(2), 193–200.Google Scholar
Krijnse Locker, J Schmid, SL (2013) Integrated electron microscopy: super-duper resolution. PLoS Biol 11(8), e1001639.Google Scholar
Kumari, S Mayor, S (2008) ARF1 is directly involved in dynamin-independent endocytosis. Nat Cell Biol 10(1), 30–41.Google Scholar
Lundmark, R, Doherty, GJ, Howes, MT, Cortese, K, Vallis, Y, Parton, RG McMahon, HT (2008) The GTPase-activating protein GRAF1 regulates the CLIC/GEEC endocytic pathway. Curr Biol 18(22), 1802–1808.Google Scholar
Mao, M, Wang, L, Chang, CC, Rothenberg, KE, Huang, J, Wang, Y, Hoffman, BD, Liton, PB Yuan, F (2017) Involvement of a Rac1-dependent macropinocytosis pathway in plasmid DNA delivery by electrotransfection. Mol Ther 25(3), 803–815.Google Scholar
Massol, RH, Boll, W, Griffin, AM Kirchhausen, T (2006) A burst of auxilin recruitment determines the onset of clathrin-coated vesicle uncoating. Proc Natl Acad Sci USA 103(27), 10265–10270.Google Scholar
Melkonyan, H, Sorg, C Klempt, M (1996) Electroporation efficiency in mammalian cells is increased by dimethyl sulfoxide (DMSO). Nucleic Acids Res 24(21), 4356–4357.Google Scholar
Mercer, J, Schelhaas, M Helenius, A (2010) Virus entry by endocytosis. Annu Rev Biochem 79, 803–833.Google Scholar
Michel, MR, Elgizoli, M, Koblet, H Kempf, C (1988) Diffusion loading conditions determine recovery of protein synthesis in electroporated P3X63Ag8 cells. Experientia 44(3), 199–203.Google Scholar
Mir, LM, Bureau, MF, Gehl, J, Rangara, R, Rouy, D, Caillaud, JM, Delaere, P, Branellec, D, Schwartz, B Scherman, D (1999) High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc Natl Acad Sci USA 96(8), 4262–4267.Google Scholar
Mukherjee, S, Ghosh, RN Maxfield, FR (1997) Endocytosis. Physiol Rev 77(3), 759–803.Google Scholar
Neu, JC Krassowska, W (1999) Asymptotic model of electroporation. Phys Rev E 59(3), 3471–3482.Google Scholar
Neumann, E, Schaefer-Ridder, M, Wang, Y Hofschneider, PH (1982) Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1(7), 841–845.Google Scholar
Nonnenmacher, M Weber, T (2011) Adeno-associated virus 2 infection requires endocytosis through the CLIC/GEEC pathway. Cell Host Microbe 10(6), 563–576.Google Scholar
Oh, P, Borgstrom, P, Witkiewicz, H, Li, Y, Borgstrom, BJ, Chrastina, A, Iwata, K, Zinn, KR, Baldwin, R, Testa, JE Schnitzer, JE (2007) Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung. Nat Biotechnol 25(3), 327–337.Google Scholar
Palade, GE Bruns, RR (1968) Structural modulations of plasmalemmal vesicles. J Cell Biol 37(3), 633–649.Google Scholar
Parton, RG Simons, K (2007) The multiple faces of caveolae. Nat Rev Mol Cell Biol 8(3), 185–194.Google Scholar
Pathak, RK, Merkle, RK, Cummings, RD, Goldstein, JL, Brown, MS Anderson, RG (1988) Immunocytochemical localization of mutant low density lipoprotein receptors that fail to reach the Golgi complex. J Cell Biol 106(6), 1831–1841.Google Scholar
Prasad, K, Barouch, W, Greene, L Eisenberg, E (1993) A protein cofactor is required for uncoating of clathrin baskets by uncoating ATPase. J Biol Chem 268(32), 23758–23761.Google Scholar
Romer, W, Berland, L, Chambon, V, Gaus, K, Windschiegl, B, Tenza, D, Aly, MR, Fraisier, V, Florent, JC, Perrais, D, Lamaze, C, Raposo, G, Steinem, C, Sens, P, Bassereau, P Johannes, L (2007) Shiga toxin induces tubular membrane invaginations for its uptake into cells. Nature 450(7170), 670–675.Google Scholar
Rosazza, C, Deschout, H, Buntz, A, Braeckmans, K, Rols, MP Zumbusch, A (2016) Endocytosis and endosomal trafficking of DNA after gene electrotransfer in vitro . Mol Ther Nucleic Acids 5, e286.Google Scholar
Rothnie, A, Clarke, AR, Kuzmic, P, Cameron, A Smith, CJ (2011) A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin. Proc Natl Acad Sci USA 108(17), 6927–6932.Google Scholar
Sabharanjak, S, Sharma, P, Parton, RG Mayor, S (2002) GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway. Dev Cell 2(4), 411–423.Google Scholar
Sesack, SR, Miner, LH Omelchenko, N (2006) Preembedding immunoelectron microscopy: applications for studies of the nervous system. In Neuroanatomical Tract-Tracing 3, Zaborszky L, Wouterlood FG and Lanciego JL (Eds.), pp. 698–765. New York, NY: Springer.Google Scholar
Shin, JS, Gao, Z Abraham, SN (2000) Involvement of cellular caveolae in bacterial entry into mast cells. Science 289(5480), 785–788.Google Scholar
Spassova, M, Tsoneva, I, Petrov, AG, Petkova, JI Neumann, E (1994) Dip patch clamp currents suggest electrodiffusive transport of the polyelectrolyte DNA through lipid bilayers. Biophys Chem 52(3), 267–274.Google Scholar
Stan, RV (2005) Structure of caveolae. Biochim Biophys Acta 1746(3), 334–348.Google Scholar
Sukharev, SI, Klenchin, VA, Serov, SM, Chernomordik, LV Chizmadzhev Yu, A (1992) Electroporation and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores. Biophys J 63(5), 1320–1327.Google Scholar
Sukumaran, SK, Quon, MJ Prasadarao, NV (2002) Escherichia coli K1 internalization via caveolae requires caveolin-1 and protein kinase Calpha interaction in human brain microvascular endothelial cells. J Biol Chem 277(52), 50716–50724.Google Scholar
Tan, X, Heureaux, J Liu, AP (2015) Cell spreading area regulates clathrin-coated pit dynamics on micropatterned substrate. Integr Biol (Camb) 7(9), 1033–1043.Google Scholar
Uehara, K Miyoshi, M (1999) Tubular invaginations with caveolae and coated pits in the sinus endothelial cells of the rat spleen. Histochem Cell Biol 112(5), 351–358.Google Scholar
van den Pol, AN (1986) Tyrosine hydroxylase immunoreactive neurons throughout the hypothalamus receive glutamate decarboxylase immunoreactive synapses: a double pre-embedding immunocytochemical study with particulate silver and HRP. J Neurosci 6(3), 877–891.Google Scholar
Wu, M Yuan, F (2011) Membrane binding of plasmid DNA and endocytic pathways are involved in electrotransfection of mammalian cells. PLoS One 6(6), e20923.Google Scholar
Xie, TD Tsong, TY (1993) Study of mechanisms of electric field-induced DNA transfection. V. Effects of DNA topology on surface binding, cell uptake, expression, and integration into host chromosomes of DNA in the mammalian cell. Biophys J 65(4), 1684–1689.Google Scholar
Zaas, DW, Duncan, MJ, Li, G, Wright, JR Abraham, SN (2005) Pseudomonas invasion of type I pneumocytes is dependent on the expression and phosphorylation of caveolin-2. J Biol Chem 280(6), 4864–4872.Google Scholar
Zaharoff, DA, Henshaw, JW, Mossop, B Yuan, F (2008) Mechanistic analysis of electroporation-induced cellular uptake of macromolecules. Exp Biol Med (Maywood) 233(1), 94–105.Google Scholar
Zeigerer, A, Gilleron, J, Bogorad, RL, Marsico, G, Nonaka, H, Seifert, S, Epstein-Barash, H, Kuchimanchi, S, Peng, CG, Ruda, VM, Del Conte-Zerial, P, Hengstler, JG, Kalaidzidis, Y, Koteliansky, V Zerial, M (2012) Rab5 is necessary for the biogenesis of the endolysosomal system in vivo . Nature 485(7399), 465–470.Google Scholar